The invention relates to inducible expression systems, to the corresponding transformed strains and to methods for obtaining them for producing proteins, in particular heterologous proteins, in yeasts.
The recombinant DNA technique enables genes for heterologous proteins to be expressed in yeasts. Thus, the construction of vectors containing yeast promoter sequences corresponding to the genes for glycolytic enzymes of Saccharomyces cerevisiae, such as 3-phosphoglycerate kinase (PGK), an alcohol dehydrogenase (ADH1) or glyceraldehyde-3-phosphate dehydrogenase (GPD), has enabled important proteins to be produced by fusion of their coding sequence to the yeast promoter, for example leukocyte interferon according to Hitzeman R. A. et al., Nature 293, 717-722 (1981) or hepatitis B surface antigen according to Bitter G. A. et al., Gene 263-274 (1984).
In many cases, it has been observed that the heterologous protein produced is toxic for the host cell, and leads to instability of the plasmids and the selection of cells which no longer express the protein in question. Methods enabling the expression of the gene for the protein to be repressed during the cellular growth phase, and then a high production of the protein to be induced during the final stage of culture, have been found using a promoter regulated by a change in carbon sources, for example the promoter of the galactokinase gene (GAL1) or the promoter of the uridinediphosphoglucose 4-epimerase gene (GAL10) which correspond to two of the four genes responsible for the utilization of galactose in yeast and whose expression is repressed by glucose and induced by galactose. Vectors comprising the GAL1 promoter linked to a heterologous gene have been described, enabling yeast to be grown in a medium containing glucose, and the protein then to be expressed when galactose is present in the medium. Yeast strains transformed with such vectors are described, for example, in British Patent Application 2,137,208, and these vectors, under the control of the GAL1 promoter, express in the presence of galactose proteins such as bovine growth hormone or prorenin. Most yeast promoters contain, upstream from TATA elements which mediate transcription initiation, elements which regulate transcription, xe2x80x9cupstream activator sequencesxe2x80x9d (designated UAS), which represent the binding site of proteins which regulate transcription, such as GAL4 for the activation of the genes of galactose metabolism, these proteins themselves being under the control of their own promoters.
Different means have already been proposed for improving the efficiency of transcription of heterologous genes in yeasts. In particular, it has been proposed to use hybrid promoters which contain, upstream from the TATA element of a constitutive yeast promoter, an extrinsic UAS regulatory sequence, such as UASg which corresponds to the GAL1-GAL10 intergenic sequence and which is inserted, for example, upstream from the TATA element of a constitutive yeast promoter, such as GPD as described in Patent Application WO 86/00,680, or such as PGK as described in European Patent Application 258,067. The inserted UASg sequences enable the constitutive promoter to be successively repressed and then expressed by the carbon source used, such as glucose as a repressor and galactose as an inducer.
However, the use of promoters in general or of any hybrid promoter involves a control over totally repressed or induced levels, which is difficult to achieve because these levels involve a group of proteins which regulate transcription. Thus, the GAL4 gene activates transcription by the presence of galactose, which is directly or indirectly responsible for the dissociation of a complex between GAL4 and an antagonistic protein GAL80, while other proteins appear to be involved in the repression by glucose, such as GAL82 and GAL83 according to Yocum R.R. et al., MBC 4 1985-1998 (1984).
In addition to the drawbacks mentioned above, this type of system based on induction with galactose and/or repression with glucose is not very versatile; in effect, it cannot be used for yeasts which do not assimilate glucose and/or galactose and cannot be used in the case where one of these components constitute all or part of the carbon substrate of the culture medium.
It is hence advantageous to have available in yeasts inducing systems capable of being induced by products which are not in themselves necessary for the culturing of these cells, which is the case, for example, with hormones, in particular steroid hormones.
Thus, the present invention relates to a method for the preparation of a protein by yeasts, according to which:
yeast cells which contain the following are cultured:
a DNA sequence coding for the said protein under the control of elements providing for its expression in yeasts, the said elements comprising a transcription control sequence which is inducible by a complex formed by a receptor and a ligand,
a DNA sequence which is functional in yeast, coding for the said receptor, the receptor comprising two essential portions, one of which recognizes the ligand so as to form a complex with the said ligand and the other binds to the said transcription control sequence; the portion of the receptor which recognizes the ligand is preferably of higher eukaryotic origin, and the ligand is not necessary for the culturing of the cells but is capable of entering the said cells when added to the culture medium,
the said ligand is added to the culture medium at an appropriate time point for induction,
the synthesized protein is recovered.
The expression of proteins by yeasts using recombinant DNA techniques is considered to be well known to those versed in the art. A considerable number of publications have already described the preparation of proteins, in particular heterologous proteins, by means of yeasts, using expression vectors. These expression vectors contain, more often than not, apart from the sequence coding for the said protein, the elements controlling its expression in yeasts, that is to say, in general, a promoter and a terminator.
In most cases, these vectors are nonintegrative vectors, that is to say plasmids; they then contain an origin of replication which is efficacious in yeasts, in particular the origin of the 2xcexc plasmid or an ars sequence peculiar to the said yeast.
These nonintegrative expression vectors contain, in addition, elements enabling provision to be made for their maintenance in the cells, either using as a selection marker a gene for resistance or alternatively an element complementing an auxotrophy of the host strain, URA3 or LEU2 for example.
In some cases, and in order to overcome the drawbacks linked to the use of self-replicating vectors, it has been possible to develop vectors providing for the integration of the sequences in question at chromosomal level. This type of vector enables a more stable strain to be obtained, but the amplification in respect of expression is sometimes smaller than with a nonintegrative plasmid.
Integration vectors contain, more often than not, at least one sequence homologous with a chromosomal sequence which will provide for exchange and integration.
It must be understood that, since the present invention relates essentially to the induction of transcription, the expression vectors which will be used can be of either the integrative or the nonintegrative type.
Although the method in question is more especially intended for the preparation of heterologous protein, it is possible to use it for expressing yeast proteins, in particular in the case of systems providing for the hyperexpression of a gene which can lead, in some cases to cell death before the optimum biomass is obtained.
xe2x80x9cHeterologous proteinxe2x80x9d is understood to mean a protein foreign to the host cell which expresses it, that is to say different in origin from that of the host cell which, according to the invention, is a yeast. The protein can be of bacterial origin, for example Escherichia coli beta-galactosidase, or of higher xc3xa9ukaryotic origin, for example of human origin, such as, for example, lymphokines, blood coagulation factors, hormones, vaccinal antigens and, generally speaking, any protein of therapeutic or industrial interest.
As has been stated, the essential feature of the invention is the combination of a transcription control sequence and a receptor complexed to a ligand. The choice of the first is conditioned by the choice of the second.
Among usable transcription control sequences, there may be mentioned, in particular, the sequences which are usually functional in higher xc3xa9ukaryotic cells, that is to say natural sequences activating the transcription of higher xc3xa9ukaryotic cells, such as, for example, an HRE (for xe2x80x9chormone responsive elementxe2x80x9d) or a variant or synthetic derivative of such a sequence, and which also fulfils in yeast this function of activation of transcription. These sequences are, in general, natural or synthetic, perfect or imperfect palindromic sequences.
Thus, for example, in the case where the transcription control sequence used is an HRE sequence, the receptor is the corresponding hormone.
As stated above, the transcription control sequence represents one of the elements providing for the expression of the desired protein in yeast. Provision for the expression in yeast may hence be made by hybrid sequences which comprise, apart from the transcription control sequence defined in the context of the invention, sequences corresponding to yeast promoter sequences. Among usable promoters, there may be mentioned the inducible promoter GAL1 referred to above, and also constitutive yeast promoters such as the PGK promoter.
In general, the TATA element of the promoter is retained and the transcription control sequence may be placed at variable distances upstream from the TATA element. It is especially advantageous to be able to have available a minimal structure for the hybrid promoter, that is to say a structure in which the distance between the TATA element and the transcription control sequence is small while remaining sufficient to provide for satisfactory inducibility.
xe2x80x9cReceptorxe2x80x9d in the sense of the invention is hence understood to mean a protein which is functional in higher xc3xa9ukaryotic cells, or variants or corresponding synthetic derivatives which display in yeast the functional properties of the native receptor.
The term xe2x80x9creceptorxe2x80x9d also comprises chimeric proteins, that is to say a structure in which the two essential portions are of different origins. For example, a preferred combination consists in preparing a hybrid receptor in which the portion which recognizes the ligand is of higher xc3xa9ukaryotic origin and the portion which binds to the transcription control sequence is of yeast origin, but it is possible to use sequences originating from any other microorganism, for example bacteria. The transcription control sequence then comprises a sequence which is functional in yeasts, that is to say a natural sequence activating transcription in yeasts. There may be mentioned, for example, the UAS of the GAL1 promoter, which is a yeast promoter.
The subject of the invention is hence the different functional combinations between a transcription control sequence, whether natural or derived, in particular, by chemical synthesis, and the receptor which can be a natural, derived or chimeric receptor. The nature of the ligand is, in turn, determined by the choice of receptor.
Among usable receptors, there should be mentioned nuclear receptors, in particular for a steroid, and other nuclear receptors, for example for retinoids or for thyroid hormones, as well as vitamin D3. The receptor for a steroid can be a natural receptor for steroid hormones, for example the estrogen receptor, the progesterone receptor or the testosterone receptor, or a variant receptor or chimeric derivative, which is functional with respect to the transcription control sequence. The steroid can be a natural steroid such as estradiol, progesterone or testosterone, or an analog or derivative, which is functional in the complex to which the transcription control sequence contained in the vector of the invention is sensitive.
The estrogen receptor can be the natural receptor (hereinafter designated hER) or a variant or chimeric derivative which is functional in the presence of estradiol with respect to the transcription control sequence contained in the vector of the invention.
More especially, the subject of the invention is the case where the essential portion of the receptor recognizing the ligand originates from the human estrogen receptor designated hERG which, compared with the receptor designated hER, has a glycine instead of a valine at position 400, the ligand then being estradiol. According to a feature of the invention, the whole of the hERG receptor may be used as the receptor.
Compared with the human estrogen receptor hER, the complementary DNA sequence of which had been published in Green S. et al. (1986) (Nature, 320, 134-139), the hERG receptor hence comprises a glycine in place of a valine at position 400. The DNA sequence and deduced amino acid sequence published in Green et al. are as follows:
It has been possible to observe that, by using this receptor instead of the hER receptor, a greater stability of the receptor was obtained, hence eliminating all risk of denaturation capable of leading to less binding of the specific ligand, in this instance estradiol. This phenomenon is expressly true in the region of 25xc2x0 C., that is to say at the temperatures at which the method for the preparation of recombinant proteins comprising an induction stage according to the invention is carried out. It is hence preferable to use this receptor, which enables a smaller quantity of estradiol to be introduced into the culture medium in order to initiate the induction.
According to an additional feature of the invention, estradiol is added at a concentration of between 2 and 50 nM, and preferably of the order of 10 nM.
The invention relates especially to the case in which the control sequence is the sequence -605 to -634 of the chicken vitellogenin gene, which is the transcription control sequence of this gene, sensitive to estradiol (designated hereinafter ERE for xe2x80x9cestrogen responsive elementxe2x80x9d). The synthesis of oligonucleotides containing this sequence or repetitions of the latter makes it possible to obtain, according to the invention, an expression vector comprising this sequence or repetitions of the latter (designated hereinafter ERE1 or ERE3).
One of the advantages of the invention is that the induction of the expression may be initiated very simply by adding to the culture medium a ligand matched to the receptor. The implementation of the induction hence becomes completely independent of the nature of the culture medium.
The appropriate time point for the introduction of the ligand can be determined easily; in practice, it will be the point at which a high level of biomass has been attained in the fermentation vat.
The invention relates most especially to the vectors, the structure and method for production of which will be given later by way of example in the experimental part.
It is appropriate to note that the elements for the expression of the protein and the elements providing for the expression of the receptor can occur on a single plasmid or on two different plasmids each containing an origin of replication which is functional in yeasts. However, it is also possible to arrange for the use of vectors providing for the integration of some of these elements, for example the production of the receptor can originate from sequences integrated on a chromosome of the cell.
Thus, the present invention also relates to the vectors carrying the sequences which are usable for the transformation of yeast cells.
The invention also encompasses the yeast strains which are usable in the method according to the invention.
As yeast strains, all strains customarily used for the production of recombinant proteins may be used. Saccharomyces strains such as S. cerevisiae may be mentioned, in particular. Special mention may be made of strains displaying one or more deficiencies in proteolytic activity, for example having the pep4 mutation. There may also be mentioned strains displaying a suppresion of the proteolytic function encoded by the PRC1 gene. The use of these strains enables the quality of the proteins obtained to be improved.